Cyanocobalt and cyanoiron complexes of cobalamin



United States Patent This invention relates to new compounds having vitamin B activity. More particularly, this invention relates to certain heavy metal cyanide complexes of cobalamin having important physical and chemical properties.

Cyanocobalamin (vitamin B is Well recognized as a vital nutritional factor in the maintenance of health and as a therapeutic agent in the correction or alleviation of various disorders and diseases. The present invention makes available compounds possessing vitamin B activity and serving, additionally, as valuable intermediates in the preparation of vitamin B and its derivatives. In particular, it has been found that the complexes of the present invention exhibit desirable stability, most notably in the presence of acids, thereby providing a means for the formulation of vitamin B with ingredients normally deleterious to the stability of vitamin B 2 such, for example, as ascorbic acid.

The novel complexes of the present invention are composed of cobalamin and iron cyanide or cobalt cyanide in the molecular proportions of two molecules of cobalamin to one molecule of heavy metal cyanide ion. The complex of cobalamin and cobalt cyanide, referred to hereinafter as cyanocobalt cobalamin, has the following properties:

LLD ACTIVITY (APPROXIMATELY 55% OF CYANOCO- BALAMIN) Ultraviolet Spectrum Elemental Analysis my i iiin The complex of cobalamin with iron cyanide, hereinafter referred to as cyano-lron cobalamin, is characterized by the following properties:

The novel complexes of the present invention are prepared biosynthetically by carrying out a fermentation procedure of the type described in US. Patent No. 2,816,856 in the presence of a single or complex cobalt or iron cyanide-containing compound, such as cobaltous cyanide,

3,115,489 Patented Dec. 24, 1963 cobaltous cobaltic cyanide, potassium ferrocyanide, potassium ferricyanide or sodium nitroprusside. The heavy metal cyanide compounds are added in such quantities as will provide a cyanide content in the range of from about 20 to about parts per million. For optimum results, it has been found that the heavy metal cyanide compounds should be added in quantities to provide a cyanide content of 50 parts per million. The addition should be effected at a pH of from about 4 to about 9, and preferably at a pH of 6.0-6.5.

The microorganisms which may be employed in the practice of this invention include those of the genus Propionibacteria which are known to produce vitamin B in the presence of a vitamin B precursor (such as 5,6- dimethylbenzirnidazole) among which are included P. arabinosum (A.T.C.C. 4965), P. thoenii (A.T.C.C. 4892), (A.T.C.C. 4871), P. zeae (A.T.C.C. 4964), P. pentasaceum (A.T.C.C. 4875) and P. petersonii (A.T.C.C. 4870); and those which produce vitamin B Without a precursor, such as P. freztdenreiclzii (A.T.C.C. 6207) and P. shermanii (A.T.C.C. 9614).

The nutrient media useful in the process of this invention include the usual sources of assimilable carbon and nitrogen. As sources of assimilable carbon, there may be used: (1) carbohydrates such as glucose, fructose, Xylose, galactose, lactose, and maltose; (2) substances containing carbohydrates such as whey, milk, cornsteep liquor, grain mashes, and molasses; (3) polyhydric alcohols such as glycerol and mannitol; (4) fats, such as lard oil, soybean oil, corn oil, butterfat and cotton seed oil; and (5) fatty acids such as acetic, propionie, pyruvic, stearic palmitic, oleic, and linoleic. Sources of available nitrogen include: (1) organic nitrogen compounds such as proteinaceous materials e.g. casein, urea, soybean meal, fish meal, yeast or yeast products, whey or whey concentrates, amino acids and liver cake; and (2) inorganic compounds such as nitrates or ammonium compounds. The nutrient media should also include metallic cations including cobalt, molybdenum, potassium, sodium, magnesium, iron, copper, manganese, the anions chloride, sulfate, phosphate, and carbonate, and the vitamins, thiamin, niacin, biotin, folic acid, pyridoxine, riboflavin, and p-aminobenzoic acid. These ions and vitamins may be present in the crude materials used in the nutrient medium. In addition, of course, the heavy metal cyanide compounds of this invention are also added as hereinbefore described. The fermentation process may be carried out at temperatures from about 20 C. to about 40 C. and may be operated under aerobic, anaerobic or microaerophillic conditions depending on the microorganism.

If the microorganism produces vitamin B without the presence of a precursor, none need be added to the broth. If a precursor is necessary it may be added to the fermentation medium either initially, or continuously or intermittently during the incubation period. After a sufficient incubation time (about one to ten days) the fermented medium may be dried and incorporated as a supplement into the feed for animals, such as chickens and pigs, or the cyanocobalamins therein may be recovered in more purified form by usual procedures.

When fermenting with a precursor requiring microorganism, the nature of the precursor employed depends on the physiologically active cobalamin desired. Thus, if vitamin B is desired, 5,fi-dimethylbenzimidazole or another vitamin B precursor, such as 2,3-dimethyl-5,6-diaminobenzene, 2,3-dinitro-5,6-dimethylbenzene or 2,3-dimethyl-4-amino-5-nitrobenzene, may be used. Thus, as summarized in the following table, the nature of the resulting cobalamin will depend on the precursor chosen. In this table, the symbol Y is that in the formula Y-cobalamin.

Product (Y-cobalamins) Product (Y-cobalamins) Precursor Precursor Name Y is Name Y S 5 p D t 1 Ti] 0H Bouzotriazole .r N\ ttenuates O methyl-5,6- cobalamiu. C 0-4311; 1 diaminobcnzene, CH ll 2,3-Dinitro-5,6- C OOH3 N dimothylbcnzene, 2,3- Dimethyl-4- 1T1 CH %$1l? ggmmo- Alternatively, the novel complexes of this invention 1 can be prepared by the reaction of hydroxo-cobalamin Benziruidazole, 1,2- Benzimidazole- N OH with an appropriate heavy metal cyanide compound. g f g gf Cobalamm This reaction is generally efiected in neutral solution at iii i h nlino-z- OH II I about room temperature. However, the use of higher mtmbenzene' temperatures is not precluded, and reactions have been N CH carried out at temperatures up to about 100 C. The I reaction period will, of course, depend upon the temperal ture employed, room temperature reactions taking place 1 l l i l l l% 5- rii ti ggl onziover a period of from one to about eighteen hours. At Tr niuliroineth'yh da C F3 higher temperatures, the reaction time will be correspondfiffiffiit cobalamm' CH g ingly decreased, periods of about 15 minutes being satismoth ylphcnylencfactory when the temperature approaches the upper diaminol,2. CH limit.

The complexes are readily convertible to cyanocobala- 1 min by treatment with potassium cyanide at either room 5-Meth0x y7-br0m0 5-Meth y-7- N or elevated temperatures. In addition, cyano-iron coifd ggol e 0 on balamin is easily converted to cyanocobalamin by treatmcthoxyphgnylcobalalnin. 01-1: II I ment with sodium hydroxide at pH 9, complete conver- C OCH3 sion being brought about within one-half hour. The N 011 conversion can be effected on the recovered, pure product I or it can be carried out on the concentrate obtained from l the fermentation broth. Quinammw /N\ 35 The following examples are illustrative of the practice of this invention: Example I I A medium containing 7.6% beet molasses, 1.5% yeast 9 autolysate solids, 2.0% protein hydrolysate, 1.0% corn 4(3IVDQumaZ0hne" o i iii i z nne 410 steep liquor, 5 ppm. cobalt (cobalt sulfite) and 2.0% CObalamm- CaCO is dispensed into 1 liter Erlenmeyer flasks at the rate of 500 ml. per flask. Eighty-two milligrams of Co(CN) '3H 0 (providing a cyanide content of 50 0 ppm.) are then added to the medium and the supplemerited medium is then sterilized by autoclaving at 121 2,4-Dichloroquin- 2,4-Dicl110r0quin- N C. for 20 minutes. After cooling, the flasks are inocuazohne' f lated With an active vegetative culture of P. freudenrez'chii N (ATCC 6207) and incubated at 30 C. on a slow rotary shaker for 96 hours. The pH is adjusted to 7.08.0 twice 1 daily with sterile NaOH. The progress of the fermenta- C1 tion is followed by periodic tests for residual sugar, and l-Acetsmido-3- 1-Acetamido-3- NIIC 0 CH3 contamination is checked by conventional plating methods. g gg 2$$fij 1 I At the conclusion of fermentation, the bacterial cells are harvested by centrifugation and the resulting cell cream extracted With an equal volume of acetone at room \N/ OCH3 temperature for 20 minutes. The acetone extract is evaporated to ,1 of its original volume, washed with chloroform and then extracted with phenol-benzene (30- 'i gg 70%). One-half volume acetone is added to the phenol GO benzene and this mixture extracted with a small volume of distilled Water. After Washing the distilled Water with N an equal volume of chloroform, the volume is reduced to l 0.2 0.5 ml. An aliquot of this rich aqueous concentrate is taken fifgg gl'j 553,3 2? g for potency determination after which 100200 microcobalamim 5 $4313 grams of pigment were applied to Whatman No. 3MM

O CH filter paper. 27 cm. of this paper is exposed to electrog phoresis in a 2 N acetic acid for 16 hours at 280 volts. Examination of the resulting electropherogram shows that I the cyanocobalt cobalamin represents about of the Quinoxaliue QIgIgZIfilIiInS-I //N\ total cobalamin pigments.

Example II A medium containing 4% clintose, 1.5% yeast autolysate solids, 2.0% protein hydrolysate, 1.0% corn steep liquor and 2.0% CaCO is inoculated with an active vegetative culture of P. freudem'eichii (ATCC 6207). Thirtyfive milligrams of Co(CN),-;-3H O (providing a cyanide content of 50 ppm.) are added to the medium and sterilization annd fermentation are then carried out as in Example I. As in Example I, the cyanocobalt cobalamin represents about 90% of the total cobalamin pigments.

Example 111 Ten gallons of a medium containing 5.4% beet molasses, 2.4% cane molasses, 1.5% corn steep liquor, 2% protein hydrolysate 0.5% yeast autolysate solids and 5 p.p.m. cobalt (cobalt sulfate) is dispensed into a fermentation tank and sterilized for minutes at 120 C. The sterilized medium is inoculated with 2 liters of a vegetative inoculum of P. freudenrez'chiz' (ATCC 6207) and fermentation is allowed to proceed for 110 hours at C. using mild aeration of 0.250.55 cubic feet per minute of air. The pH of the medium is controlled during fermentation between 6.0 and 6.6. Cobaltous cyanide Co(CN) 3H O is added intermittently during the fermentation at a level providing a total cyanide content addition of 170 p.p.m. The electrophoresis method described in Example I shows that the cyanocobalt cobalamin represents about 95% of the total cobalamin pigments.

Example IV Following the procedure of Example I, but substituting K Fe(CN) -3H O for CO(CN) -3H O there is obtained the corresponding complex cobalamin and iron cyanide (cyano-iron cobalamin).

Example V 153 mg. of crystalline hydroxocobalamin is added to 25 cc. of an aqueous solution containing 300 mg. of cobaltous cyanide. The solution is allowed to stand for 16 hours at room temperature and then extracted with 12 cc. of 88% (aqueous) phenol. The resulting organic phase is separated and washed three times with 10 cc. portions of water, each of which contains 3.2% phenol. To the phenol layer, there is added 24 cc. of benzene, cc. of acetone and 5 cc. of Water. The resulting aqueous phase is separated, washed with three 5 cc. portions of chloroform and then lyophilized. The resulting amorphous residue is purified by continuous paper electrophoresis in 2 N acetic acid at 280 volts. The fractions containing the cyano-cobalt cobalamin are combined, extracted with 10 cc. of phenol and the phenol extract washed with three 10 cc. portions of water, each containing 3.2% phenol. To the phenol layer, there is added 24 cc. of benzene. 35 cc. of acetone and 5 cc. of water. The resulting aqueous phase is separated, washed with three 5 cc. portions of chloroform and then lyophilized. The resulting amorphous product is cyanocobalt cobalamin, identical with the product obtained according to the procedure of Example I.

Example VI To 25 cc. of water containing 300 mg. of potassium fer rocyanide trihydrate, there is added 152 mg. of crystal line hydroxocobalamin. The resulting solution is allowed to stand for 1 6 hours at room temperature, and the product is then isolated and purified by following the procedure detailed in Example V. The product obtained is cyano-iron cobalamin, identical with the product obtained according to Example IV.

Example VII To 25 cc. of water containing 300 mg. of sodium nitroprusside, there is added 152 mg. of crystalline hydroxocobalamin. The resulting solution is allowed to stand for 16 hours at room temperature, and the product is then isolated and purified by following the procedure detailed in Example V. The product exhibits an additional maximum at 5.23;, characteristic of the nitroso group.

The invention may be variously otherwise embodied within the scope of the appended claims.

What is claimed is:

1. Cyanocobalt cobalarnin, a complex of cobalamin and cobalt cyanide in the molecular proportion of two molecules of cobalamin to one molecule of cobalt cyanide ion, said complex having an ultraviolet spectrum containing peaks at the following wavelengths: 273, 286, 356, 408, 505, 535 millirnicrons, and an elemental analysis of carbon, 52.45%; hydrogen, 6.05%; nitrogen, 12.92%; cobalt, 6.33%, and phosphorus, 1.98%

2. Cyanoiron cobalamin, a complex of cobalamin and iron cyanide in the molecular proportion of two molecules of cobalamin to one molecule of iron cyanide ion, said complex having an ultraviolet spectrum containing peaks at the following Wavelengths: 356, 506 and 535 millimicrons, and an elemental analysis of carbon, 54.12%; hydrogen, 6.31%, nitrogen, 12.72%, cobalt, 4.25%, iron, 1.98%, and phosphorus, 2.112%.

References Cited in the file of this patent UNITED STATES PATENTS 2,650,896 McDaniel et a1 Sept. 1, 1953 2,874,089 Zuck Feb. 17, 1959 2,879,203 Barthelemy et al Mar. 24, 1959 2,971,891 Cords et al. Feb. 14. 1961 

1. CYANCOBALT COBALAMIN, A COMPLEX OF COBALLAMIN AND COBALT CYANIDE IN THE MOLECULAR PROPORTION OF TWO MOLECULES OF COBALAMIN TO ONE MOLECULE OF COBALT CYANIDE ION, SAID COMPLEX HAVING AN ULTRVIOLET SPECTRUM CONTAINING PEAKS AT THE FOLLOWING WAVELENGTHS: 273, 286, 356, 408, 505, 535 MILLIMICRONS, AND AN ELEMENTRAL ANALYSIS OF CARBON, 52.45%; HYDROGEN, 6.05%; NITROGEN, 12.92%; COBALT, 6.33%, AND PHOSPHOROUS, 1.98% 